This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Infectious diseases remain one of the leading causes of death worldwide, emphasizing the limitations of current treatments and the need for novel approaches. In recent years, it has become evident that bacterial cultures also exert virulence in infections as multi-cellular communities, so-called biofilms. The majority of chronic infections can be attributed to biofilms, increasing their pathogenicity and contributing to the spreading of antibiotic tolerance and resistance. The formation of biofilms involves signaling processes controlling the switch from a free-swimming, planktonic state to a surface-attached, self-contained social life form. Although biofilm-specific genes have been identified and the process has been studied morphologically, how biofilm formation is regulated, and the underlying signaling mechanisms involved, are largely unknown. We study a second messenger unique in the microbial world, cyclic di-GMP, that recently has been shown to control secretion, cell adhesion and motility leading to biofilm formation and increased cytotoxicity. The protein domains responsible for catalyzing the synthesis and degradation of cyclic di-GMP, namely GGDEF and EAL domains, respectively, have been identified in large numbers in almost every eubacterial genome sequenced to date. GGDEF and EAL domains occur in the context of diverse multi-domain signaling proteins, and as part of multi-component signaling systems, thus suggesting a role in sensing environmental cues. We study the enzymatic and regulatory mechanisms of these key switches in biofilm formation and cytotoxicity, using structural and functional approaches. Given the absence of c-di-GMP in eukaryotic cells, we envision that these signaling systems might provide attractive targets for the development of novel antibacterials and methods to modulate biofilm formation.